重组载体pLNCX/iNOS的构建及其在NIH3T3细胞中滴度的测定-东南大学学报医学版

重组载体pLNCX/iNOS的构建及其在NIH3T3细胞中滴度的测定

单位：1.东南大学附属中大医院,血液科,江苏,南京,210009； 2.江苏省肿瘤研究所,江苏,南京,210009； 3.南京军区军事医学研究所,江苏,南京,210004

分类号：Q781, Q782, Q784, Q786

出版年·卷·期（页码）：2002·21·第二期（137-139）

摘要：

目的:构建重组质粒pLNCX/iNOS.方法:用PCR技术从pCMV/iNOS质粒中扩增出iNOS全长cDNA,与逆转录病毒pLNCX构建重组质粒pLNCX/iNOS;通过脂质体介导把重组质粒转染入包装细胞PA317中,经G418筛选出抗性克隆.通过NIH3T3细胞测定病毒滴度.结果:经过酶切分析和核苷酸序列分析鉴定重组质粒构建正确.G418筛选出16个抗性克隆,能稳定合成并分泌重组逆转录病毒颗粒.NIH3T3细胞测定病毒滴度为2.1×104CFUml-1.结论:逆转录病毒载体pLNCX可有效地介导iNOS的转染.

Objective To construct the recombinant pLNCX/iNOS.Methods Full length cDNA of iNOS were amplified by PCR from pCMV/iNOS vector with retroviral vectors to construct pLNCXiNOS.Purified recombinant retroviral vectors DNA were transferred into the amphotropic packaging cell line PA317 by means of liposome mediated method and screened anti?G418 positive clones which were cultured?abstracted superant liquid for further concentration.The viral titer was determined with the NIH3T3.Results The gene rank of iNOScDNA amplified by PCR was coincidence with its gene map through enzyme cutting and gene sequencing analysis.16 anti?G418 positive clones can stably synthesis and excrete recombinant retroviral vectors.The titer of recombinant retroviral vector was 2.1×10 4CFU·ml -1 .Conclusion The iNOS gene can be effectively transferred by retroviral vector pLNCX.

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